Process for micro biological production of proteins

ABSTRACT

The invention is based on the finding that furin belongs to a family of endoproteolytically active enzymes and relates to a process in the in vitro cleavage of a protein by treating the protein in the presence of Ca 2+   ions with furin, or an endoproteolytically active fragment, derivative or fusion protein of furin. The invention can be used for the (micro) biological production of a protein by culturing genetically engineered cells expressing a pro-form of the protein as well as furin and isolating the protein formed. The invention also relates to a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents or adjuvants, as well as an endoproteolytically active amount of furin, or a fragment or derivative of furin having an endoproteolytic activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 08/568,152, filed Dec. 6,1995, now abandoned, which is a divisional of application Ser. No.07/849,420, filed Jun. 24, 1992, which was the national stage ofInternational Application No. PCT/NL90/00151, filed Oct. 12, 1990.

The invention relates to both a pharmaceutical composition having anendoproteolytic activity and a process for the (micro)biologicalproduction of a protein and for the in vitro cleavage of a protein, inparticular a precursor protein by processing the protein with anendoproteolytically active enzyme.

The invention described herein is the result of a further study into thepossible physiological significance of a furin, a human proteindescribed in European patent application EP-A-0 246 709, which is theexpression product of the fur gene located in the genome upstream of thehuman fes/fps protooncogene. The patent application referred to andother publications by the same research group (Roebroek et al., Molec.Biol. Rep. 11, 1986, 117-125; Roebroek et al., EMBO J. 5, 1986,2197-2202; and Schalken et al., J. Clin. Invest. 80, 1987, 1545-1549)show that on the basis of the limited DNA data then available, it wasimpossible to determine the function of the product of the fur gene.What could be determined was that the furin is probably amembrane-associated protein which has a function in which certainrecognition structures play a role. It was also observed at the timethat the fur gene is expressed as a 4.5 kb mRNA in liver, kidney,spleen, thymus and brain, whereas the expression in lung tissue is veryslight; in non-small-cell lung carcinomas, on the other hand, a highlyincreased expression was found to occur, on the ground of which the furgene was suggested to have a utility as a tumor marker.

Within the framework of the above research, the complete nucleotidesequence of a genomic DNA fragment of about 21 kbp containing the furgene has meanwhile been determined (Van den Ouweland et al., Nucl. AcidsRes. 17, 1989, 7101-7102), while the nucleotide sequence of thecorresponding fur cDNA has also been determined (Van den Ouweland etal., Nucl. Acids Res. 18, 1990, 664). On the basis thereof it is nowpossible for the fur gene to be completely characterized, at both thelevel of genomic organization structure and the level of the encodingsequences. From these encoding nucleotides sequences, the amino acidsequence of the furin can also be derived.

SUMMARY OF THE INVENTION

A computer analysis of this amino acid sequence has now surprisinglyrevealed that furin is highly similar to subtilisin-like proteases asencoded in yeast by the KEX1 gene of Kluyveromyces lactis and the KEX2gene of Saccharomyces cerevisiae, and that furin is evidently thehigher-eukaryotic form (found in Man and in animals, such as monkey,cat, rat, mouse, chicken and Drosophila) of these endoproteases. Morespecifically it has been found that the furin exhibits a certain degreeof homology with the catalytic domain of the hitherto describedbacterial subtilisins (about 20 enzymes), such as thermitase ofThermoactinomyces vulgaris and subtilisin BPN' of Bacillusamyloliquefaciens, and exhibits a striking high homology withsubtilisin-like proteases, such as the expression product of the KEX1gene of the yeast Kluyveromyces lactis and the expression product of theKEX2 gene of the yeast Saccharomyces cerevisiae. The furin, whichcontains 794 amino acids, exhibits in the domain of the amino acids 97to 577 an overall homology of about 80.0% with the amino acids 123-584of the expression product of said KEX1 gene (i.e., 41.6% identical aminoacids and 38.3% conservative substitutes) and an overall homology ofabout 78.9% with the amino acids 134-597 of the expression product ofsaid KEX2 gene (i.e., 39.4% identical amino acids and 39.5% conservativesubstitutes). These amino acid regions of the yeast proteases comprisethe subtilisin-like catalytic domains. The subtilisin-like domain offurin is situated in an amino-terminal furin fragment comprising theamino acids 108-464.

With regard to the subtilisin-like proteases, reference is made to thefollowing publications: Tanguy-Rougeau et al., FEBS Letters 234, 1988,464-470; Mizuno et al., Biochem. Biophys. Res Commun. 156, 1988,246-254; Meloun et al. FEBS Letters 183, 1985, 195-200; Marklan et al.,J. Biol. Chem. 242, 1967, 5198-5211; Mizuno et al., Biochem. Biophys.Res. Commun. 159, 1989, 305-311; Bathurst et al., Science 235, 1987,348-350; Thomas et al., Science 241, 1988, 226-230; Foster et al.,Biochemistry 29, 1990, 347-354; Fuller et al., PNAS USA 88, 1989,1434-1438; Julius et al., Cell 37, 1984, 1075-1089; Bourbonnais et al.,J. Biol. Chem. 263, 1988, 15342-15347; Cosman et al., Dev. Biol. Stand.69, 1988, 9-13, Schubert Wright et al., Nature 221, 1969, 235-242;Cunningham et al., Yeast 5, 1989, 25-33; Davidson et al., Nature 333,1988, 93-96.

As shown by the above publications, it is especially the expressionproduct of the KEX2 gene of the yeast species Saccharomyces cerevisiaewhich has been well studied and characterized. It is amembrane-associated, calcium ions dependent endopeptidase with an enzymespecificity for paired basic amino acid residues; substrate proteins arecleaved at the carboxyl site of pairs of basic amino acids containingarginine by this enzyme, which is to be defined here as a "restriction"endopeptidase (by analogy to the nomenclature in restrictionendonucleases in which a given nucleotide sequence is determinative ofthe cleavage of the DNA). The location of the enzyme is probably in astructure of the Golgi complex. The subtilisin-like domain and the Ca²⁺activation sequences are in the aminoterminal part of the protein. Inthe yeast Saccharomyces cerevisiae, the endopeptidase is involved in theproteolytic processing of precursors of killer toxin and pairingpheromone alpha factor, i.e., of pro-killer toxin and pro-alpha factor.Furthermore, the endopeptidase is found to be capable of correctlycleaving the mouse neuroendocine-peptide precursorprepro-opiomelanocortin after introduction into certain mutant mammaliancell lines with disturbed proteolytic processing, and to be capable ofprocessing proalbumin to mature albumin and to be capable of processingthe precursor of the plasma C protein.

On the ground of the established similarities between the above knownendopeptidases and the furin, it is postulated that the furin is arestriction endopeptidase which can be used for the processing ofproteins, more specifically the processing of precursor proteins ofpolypeptide hormones, growth factors, toxins, enzymes, or other types ofbiologically relevant proteins. In this connection, in vitroapplications are conceivable on the one hand, and in vivo applicationson the other, including an application within the framework of atherapeutic treatment. For such applications, the human furin may bemore suitable than the above known endopeptidases of non-human origin,or more generally an animal "furin" may be more suitable than anendopeptidase from lower organisms. The same applies to analogues orrelatives of furin not yet isolated, referred to herein as furin-likeenzymes, belonging to a larger family of restriction-endoproteolyticenzymes of which furin is the first-found representative. The variousmembers of this family will exhibit a high structural resemblance,although the sequence homology may be quite low, possibly as low asbelow 50% homology. Within this family, it will be possible todistinguish several enzyme classes, such as a group of furin-likeenzymes involved in the processing of constitutively secreted proteinsand a group of furin-like enzymes involved in the processing of proteinswhose secretion is regulated (secretion through secretory granula). Itis possible that each of these furin-like enzymes is characteristicallyexpressed in a limited number of cell types in which the enzyme isactive as a processing enzym. A limited degree of overlap between thecell and tissue distribution of these enzymes is also conceivable andcould very well be responsible for the known phenomenon of celltype-dependent differential processing of precursors.

The pituitary proteins PC1 and PC2, described recently by Seidah et al.,DNA and Cell Biol. 9, 1990, 415-424, constitute examples of suchfurin-like enzymes.

Through recombinant DNA techniques, it is possible to obtain largequantities of the protein furin. In prokaryotes, the fur gene can beexpressed as a fusion protein with beta-galactosidase (pUR vectorsystem) or the anthranilate synthetase (pATH vector system). Anotherpossibility is the synthesis of the fusion proteinglutathion-S-transferase-furin (pGEX). The advantage of this approach isthat the furin can be split off by means of thrombin. The furin can alsobe synthesized as such in prokaryotes by placing the cDNA in the correctmanner behind a suitable promotor. The pUR and pATH vector systems havebeen described in the European patent application referred tohereinbefore. pGEX is commercially available. Using the strong SV40promotor, the fur cDNA can be expressed in suitable eukaryotic cells. Inconnection with glycosylation of the protein, this approach is preferredfor certain purposes.

Furin can be purified by standard biochemical techniques in the presenceof protease inhibitors. Furin is active in a relatively acidic mediumwith a pH of 5.5, as occurs in secretory granula, but the proteinmaintains its activity also at pH 7.5. By virtue of this, a 0.2 M sodiumacetate buffer (pH 5.5) or Tris-HCl buffer (7.0) may be used in vitro.The activity of the enzyme furin depends on the presence of Ca²⁺ ions.For the in vitro enzyme activity, a calcium concentration of 2-5 mM hasbeen found to be optimal. The presence of metal chelators such as EDTAwill greatly inhibit the activity of furin. Furthermore, the presence ofheavy metal ions such as Zn²⁺, Hg²⁺ and Cu²⁺ should be avoided. Thesubstance o-phenanthrolin binds heavy metals except Ca²⁺ and thus has noadverse effect on the enzymatic activity of furin. Low concentrations ofphenyl methyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphate(DFP) up to 5 mM have no inhibitory effect. At higher concentrations ofPMSF, the enzyme function is inhibited. An in vitro incubation for twohours at 37° C. is sufficient for the processing of the protein to becleaved.

Furin can be used for the endoproteolytic processing of variousproteins. This makes it possible, for example, for in vitro producedprecursor proteins to be specifically cleaved to form biologicallyactive compositions which may be used as additional agents for thetreatment of diseases in which the precursors are not split or to aninsufficient degree. Generally speaking, furin may be said to besuitable in the processing of biologically relevant proteins.

The protein furin may also find application as a medicament, so thatpatients deficient in an endoprotease may be treated by administeringfurin, so that an adequate processing of precursor proteins is yetpossible. As a result, the cleavage products may perform their function,and it will be possible for any disturbingly high levels of precursorproteins to be reduced.

Furin is possibly also applicable for clearing depositions withsubstrate proteins in, for example, the blood circulation system, sothat obstructions of vital organs may be remedied by the administrationof furin.

Furin is further also applicable in the commercial production of allsorts of biologically active substances (e.g., other enzymes) ifprocessing is a production step therein.

The invention relates in the first place to a pharmaceutical compositioncomprising one or more pharmaceutically acceptable carriers, diluents oradjuvants, as well as an endoproteolytically active amount of furin or afurin-like enzyme, or a fragment or derivative of furin or furin-likeenzyme having an endoproteolytic activity.

The proteolytic activity is maintained when the carboxy-terminal regionwith the transmembrane domain therein has been split off. Instead of thecomplete furin or furin-like enzyme, therefore, according to theinvention, use can be made of a fragment of the enzyme which stillcontains the part responsible for the proteolytic activity. One suitablefragment is, for example, the furin fragment consisting of amino acids108-464.

The activity of the furin or furin-like enzyme, or of anendoproteolytically active fragment thereof, can further be manipulatedby introducing mutations. The invention accordingly also extends toderivatives of furin or furin-like enzyme still having endoproteolyticactivity.

According to a preferred embodiment according to the invention of such apharmaceutical composition, the furin or the furin-like enzyme, or thefragment or derivative of furin or furin-like enzyme havingendoproteolytic activity, used in the composition, has been obtainedfrom prokaryotic or eukaryotic cells which through genetic engineeringwith recombinant DNA or RNA have acquired the ability of expressing thefurin, furin-like enzyme, fragment or derivative of furin or furin-likeenzyme, whether or not in the form of a fusion protein, while in casethe furin, furin-like enzyme, fragment or derivative of furin orfurin-like enzyme is produced by the cells as a fusion protein, thefusion protein has been processed to split off the furin, furin-likeenzyme, fragment or derivative of furin or furin-like enzyme from thefusion protein.

Another possibility, however, is for the source of the furin orfurin-like enzyme used to be cells which by nature are capable ofproducing the furin or furin-like enzyme, for example, a suitable tumorcell line.

A particularly preferred embodiment of the invention concerns apharmaceutical composition containing furin itself.

An alternative particularly preferred embodiment of the inventionconcerns a pharmaceutical composition comprising an aminoterminalfragment of furin comprising at least the amino acids 108-464 of furin.

The invention further relates to a process for the in vitro cleavage ofa protein by treating the protein with an endoproteolytically activeenzyme, in which, in accordance with the present invention, the proteinis treated in the presence of Ca²⁺ ions with furin or a furin-likeenzyme, or an endoproteolytically active fragment, derivative or fusionprotein of furin or furin-like enzyme as the endoproteolytically activeenzyme.

The treatment will commonly be carried out at physiologically occurringpH and temperature values, i.e., at a pH within the range of 4-9 and ata temperature of about 37° C.

Preferably, the treatment is carried out at a pH of 5-7.5, morepreferably 5.5-7.0.

Also, according to the invention, it is preferable for the treatment tobe carried out at a temperature of 20-50° C., more preferably 30-40° C.

Furthermore, according to the invention, it is preferable for thetreatment to be carried out at a calcium concentration of 1-10 mM, morepreferably 2-5 mM.

According to a particularly preferred embodiment of the invention, thetreatment is carried out in the presence of o-phenanthrolin or anequivalent agent for binding heavy metals other than calcium.

The process according to the invention comprises a treatment ofsubstrate to be processed as such with furin (or with a furin-likeenzyme) as such, i.e., furin in an isolated or purified form, but alsocomprises a treatment with or within cells, in particular geneticallyengineered mammalian cells in which furin is expressed. Preferably,these are carefully selected, genetically engineered mammalian cells(such as COS-1 cells, CHO cells and endothelial cells) with high levelsof expression of both the fur gene and a gene encoding for the substrateto be processed. As well known to those skilled in the art, a greatlyenhanced expression can be realized by gene amplification or by usingstrong promoters. The invention even extends to applications involvingtransgenic animals, and is therefore not limited to in vitro proteinproduction and protein cleaving processes. The invention accordinglyalso comprises mammalian cells and mammals comprising DNA originatingfrom recombinant DNA encoding for furin or furin-like enzyme, andcapable of expressing the furin or furin-like enzyme. Endothelial cells,for example, are particularly ideal as mammalian cells for transport oftherapeutic genes and gene products through the body by reason of theirdistribution throughout the entire body (at the surface of bloodvessels, lung tissue, and the like) and by virtue of their interactionwith all sorts of components in the circulation of body fluids (thebloodstream and the like). Thus endothelial cells of a patient sufferingfrom some disease resulting from a disturbance in the processing ofpro-proteins, could be genetically engineered after being isolated fromthe body to remedy the defect by introducing an active fur gene, andsubsequently the genetically modified cells could be re-transplantedinto the patient. Such a gene therapy, however, is not limited toendothelial cells.

A preferred embodiment of the invention consists in a process for the(micro)biological production of a protein by culturing geneticallyengineered cells expressing a pro-form of the protein as well as furin,and possibly isolating the protein formed. For this purpose bothprokaryotic and eukaryotic cells can be used, but cells of highereukaryotes are preferred. For example, yeast cells or still better plantcells can be used. It is, however, particularly preferable to usegenetically engineered mammalian cells.

The expression "pro-form" means a form of the protein which should ormay be converted into the desired protein by processing. It may be anatural pro-form or prepro-form of the protein, but also a syntheticpro-form which is the result of a recombinant DNA construct in which thegene coding for the desired protein is preceded by an added signal orleader sequence.

As regards the substrates to be processed, generally speaking proteinswith paired basic amino acid residues can serve as a substrate. Theadditional presence of a basic amino acid residue in the -4 positionrelative to the cleavage site (i.e., 4 positions before the cleavagesite) will lead to higher efficiency. The following examples arementioned as possible substrates for processing by furin withoutcompleteness being pretended: precursors encoded by the transforminggrowth factor β (TGF-β) gene family of growth and differentiationfactors (such as TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, activiv,inhibin, Xenopus laevis Vg1 gene product, Mullerian Inhibiting Substance(MIS), decapentapeptide gene complex of Drosophila embryos and bonemorphogenetic protein, see Sporn and Roberts, Anal. N.Y. Acad. Sci. 593,1990, 1-6), precursors of growth factors, such as β-Nerve Growth Factor(β-NGF) and insulin, precursors of clotting factors, such as vonWillebrand Factor, Protein C, Factor IX and Factor X, hormones andneuropeptides, such s Proopiomelanocortin, Proenkephalin, Prodynorphin,Provasopressin, Prooxytocin, ProCRF (corticotropin releasing factor),ProGRF (growth hormone releasing factor), Prosomatostatin, Proglucagon,Procalcitonin, ProCGRP (calcitonin gene-related peptide), ProVIP(vasoactive intestinal peptide), Procaerulin and ProELH (egg layinghormone), interleukins, interferons, and hematopoietic factors.

The invention can also be applied to proteins which do not by themselvesrequire endoproteolytic processing. Examples are gene constructs inwhich, for reasons of good processing (glycosylation) or readypurification (secretion) a sequence encoding for the desired protein iscoupled to a suitable signal sequence, as has been proposed earlier, forexample, for the production of erythropoietin in yeast cells by Elliottet al., Gene 79, 1989, 167-180. In that publication, a gene construct isdescribed from the leader region of prepro-alpha factor placed beforethe erythropietin sequence. The processing of the resulting syntheticprecursor is effected in the yeast cells by the KEX2 gene productpresent therein.

BRIEF DESCRIPTION OF THE DRAWINGS

A further illustration of the invention will be given with reference tothe accompanying drawings, in which

FIG. 1 shows the amino acid sequence (in the one-letter code) of thefurin consisting of 794 amino acids (SEQ ID NO:2);

FIG. 2 shows diagrammatically the furin gene, cDNA and protein.

a. Genomic organization of part of the fur gene. Exon 1 (about 120 bp)is located at 7.2 kb upstream of exon 2. The asterisk above exon 2indicates the position of the initiation codon, and the arrow head aboveexon 16 the stop codon. Non-coding sequences are represented by blackboxes. B=BamHI; E=EcoRI; K=KpnI; S=SalI; P=PstI; X=XBaI.

b. Schematic distribution of exons in the cDNA of fur.

c. The putative localization of the various protein domains in furin.The largest exon (exon 16) encodes nearly the entire Cys-rich domain,the transmembrane domain and the cytoplasmic domain. The exons 2-12encode the presumptive prepro and catalytic domains, with codons for theactive site residues Asp46 (D), His87 (H) and Ser261 (S) in exons 5, 7and 10, respectively. This intron/exon distribution is of the samedegree of complexity as observed in the trypsin family of serineproteases. Vertical arrows indicate pairs of basic residues (Arg-Arg,Lys-Arg) which are potential autoprocessing sites; the pairs of basicamino acid residues Arg310-Lys311 and Arg341-Lys342 are possiblyinvolved in proteolytic cleavage. The N-terminus of the mature proteinis assumed to being at amino acid residue 108 directly behind thetriplet of potential cleavage sites (Lys-Arg-Arg-Thr-Lys-Arg) (SEQ IDNO:1) because an arginine residue (Arg104) at the -4 position relativeto the proposed cleavage site has been found to enhance cleavageefficiency.

The regions in which the amino acid sequences of furin, Kex1(Kluyveromyces lactis) and Kex2 (Saccharomyces cerevisiae) exhibitsimilarity include parts of the prepro domain, the entire catalyticdomain (47% identity in 322 residues) and the entire middle domain(26-31% identity in 138 residues). There is no significant similarity inthe transmembrane and cytoplasmic domains, while the Cys-rich domain isnot present in the two yeast proteins.

FIG. 3A and FIG. 3B contain a comparison of the amino acid sequences of(hfur) human furin (SEQ ID NO:3), (Kex1) Kex 1 protease (SEQ ID NO:4),(Kex2) Kex2 protease (SEQ ID NO:5), (ther) thermitase (SEQ ID NO:6),(subC) subtilisin Carlsberg (SEQ ID NO:7), and (subB) subtilisin BPN'(SEQ ID NO:8).

On the right-hand side, the numbering of the amino acid residues fromthe putative N-terminus of the mature enzymes is given, and for furinalso along the top. Probably, furin has a prepro segment of 107 residuesterminating with the sequence Lys-Arg-Arg-Thr-Lys-Arg (SEQ ID NO:1),which has three potential cleavage sites for auto-activation.

(∇)identical residues and (.) conservative substitutions in all sixsequences: (|) identical residues in at least four sequences. Pairs ofbasic residues in furin, Kex1 and Kex2 are indicated in lower caseletters. The sequence alignment is taken from a multiple alignment ofmore than 20 members of the subtilisin family of serine proteases and asuperposition of the three-dimensional structures of thermitase,subtilisin Carlsberg and subtilisin BPN' determined by X-raycrystallography. This superposition of three-dimensional structuresleads to an extended consensus core, as shown by solid bars, withdistances between topologically equivalent Cα atoms of less than 1.5 Å.Secondary structural elements common to all three proteins are indicatedas (α) α-helix, (β) β-sheet, (t) β-turn and (s) bend.

Residues known to be involved in substrate or inhibitor binding inthermitase, subtilisin Carlsberg and subtilisin BPN' through main-chainor side-chain interactions are marked with asterisk. Essential residuesof the active site (D, H and S) and of the oxyanion hole (N) areunderlined. The loops corresponding to the strongest Ca ion bindingsites in thermitase are indicated by <==Ca==>.

Boundaries of exons encoding sequences of the presumptive catalyticdomain of furin are located behind residues 17, 60, 86, 115, 173, 244,278 311 and 352.

FIG. 4 shows a schematic model of the catalytic domain of furin. Themodel is based on a ribbon drawing of subtilisin. The active site,consisting of the residues Asp46, His87 and Ser261, is situated at thetop centre. The C-terminal extension (dashed) containing additionaldomains begins at the opposite side of the catalytic domain.

Predicted positions of 8 short inserts (solid black), including anextended N terminus, relative to subtilisin, are seen to be situated insurface loops and in connections between conserved α-helix and β-sheetsecondary structural elements.

Predicted positions of two stabilizing calcium ions, Ca1 and Ca2 as inthermitase, are indicated by hatched spheres in the external loops98-105 and 68-77, respectively. All of the side chain carboxyl groupsrequired for the coordination of these two calcium ions as in thermitaseare also present in furin in topologically equivalent residues in theseloops; in addition, Asp8 and Asp55 are present to coordinate Ca1 as inthermitase and subtilisin, where in topologically equivalent positionseither Gln or Asp are the ligands.

Predicted disulfide bridges Cys104-Cys253 and Cys196-Cys226 (orCys198-Cys226) are shown in dotted lines.

Negatively charged side chain groups on the substrate binding site (top)of the furin molecule are shown as forked stalks and correspond toresidues 46, 47, 84, 121, 123, 126, 150, 151, 152, 192, 194, 199, 241,248 and 255. Most of these charges are not present in equivalentpositions in subtilisins and thermitase. Many of these negativelycharged residues could interact directly with paired basic residues inthe substrate, as they are probably located in or near the P1 and P2binding pockets for lysine and/or arginine.

The model described for the catalytic domain of furin also applies tothe Kex1 and Kex2 proteases since essentially all of the importantelements described above are present in all three proteins.

FIG. 5 shows diagrammatically the structural organization of prepro-vWFof wild type von Willebrand Factor (top part) and prepro-vWFgly763 ofthe mutant vWFgly763 (lower part). Internal homologous domains areindicated by open boxes; A1, A2 and A3 represent a triplicated domain; Bembodies the homologous domains B1, B2 and B3; C1 and C2 represent aduplicated domain; D1, D2, D3 and D4 represent four repeated domains andD' represents a partly duplicated domain. The solid line indicates theremaining amino acid sequences. The aminoterminal part contains a signalpeptide of 22 amino acid residues. The cleavage site after arginineresidue at position 763, which consists of a pair of basic amino acidresidues, is marked with an arrow. The nucleotide sequence of the DNAregion around the cleavage site of prepro-vWF is given (SEQ ID NO:10),and the deduced amino acid sequence presented in one-letter notation(SEQ ID NO:9). The nucleotide sequence of the DNA region around thecleavage site of prepro-vWFgly763 is given (SEQ ID NO:11), and thededuced amino acid sequence presented in one letter notation (SEQ IDNO:12). The point mutation in pro-vWFgly 763 (SEQ ID NO:11) is markedwith an asterisk.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

There will now be given an example of a process according to theinvention in which the endoproteolytic activity of furin is used in theprocessing of the precursor of the von Willebrand Factor (pro-vWF) as asubstrate. With regard to the structure of prepro, pro and mature vWF,reference is made to Verweij et al., EMBO J. 5, 1986, 1839-1847, andVerweij et al., J. Biol. Chem. 263, 1988, 7921-7924. Pro-vWF consists ofa pro-polypeptide (741 amino acid residues) and, at the C terminus,mature vWF (2050 amino acid residues). As explained in the abovepublications, mature vWF is formed from pro-vWF by proteolyticprocessing next to the paired basic amino acids Lys762-Arg763, and COS-1cells are a suitable host for the synthesis of constitutively secretedvWF after transfection of full-length prepro-vWF cDNA. The activity offurin in endoproteolytic processing was tested for both pro-vWF and themutant pro-vWFgly763 described by Voorberg et al., EMBO J. 9, 1990,797-803. The DNA coding for this mutant pro-vWFgly763 contains aguanosine instead of an adenosine in the 2407 position of full-lengthprepro-vWF cDNA. As a result of this mutation, the cleavage siteLys762-Arg763 of the propolypeptide is replaced by Lys762-Gly763 in thepro-vWF precursor protein mutant.

EXAMPLES Identification of Translation Products Encoded by Full Lengthfur cDNA Transfected into COS-1 Cells

To further characterize the fur gene product furin, experiments wereperformed to synthesize this protein in eukaryotic cells under thecontrol of the SV40 late promoter and to use this material in anapproach to elucidate its function. To identify the translation productsof the fur gene, an immunological approach was selected. A polyclonalantiserum raised in rabbits to a recombinant furin hybrid protein asdescribed in "Materials and methods" was used. In Western blot analysisof proteins in total lysates of bacteria transformed with pMJ109, pMJ119or pEW1 DNA, the polyclonal antiserum recognized β-gal-Δfurin1,336trpE-AS-Δfurin1 and GST-Δfurin2, respectively. In controlexperiments, the antiserum did not react with the trpE-encodedpolypeptide chain of anthralinate synthetase orglutathion-S-transferase. Using this antiserum, Western blot analysiswas performed to detect fur gene-encoded proteins in COS-1 cellstransfected with pSVLfur. Based upon nucleotide sequence data of furcDNA, synthesis of a primary translation product with a calculatedmolecular weight of 87 kDa may be expected. Two proteins with apparentmolecular weights of about 90 kDa and 100 kDa, respectively, weredetected in transfected COS-1 cells as compared to non-transfected COS-1cells (results not shown).

The presence of two forms of furin in COS-1 cells transfected withpSVLfur DNA indicates that furin is subject to post-translationalmodification. It is possible, that the 100 kDa protein is a glycosylatedform of the primary product of about 90 kDa. However, it is alsotempting to speculate that the 100 kDa polypeptide would represent thepro-form, while the 90 kDa polypeptide represents mature furin,generated by proteolytic (auto)processing of the peptide bond betweenresidues 107 and 108.

It is noted that non-transfected COS-1 cells also contain small amountsof immunoreactive material with apparent molecular weights of 90, 60 and40 kDa. Although the identity of these proteins remains to beestablished, it is conceivable that the 90 kDa protein represents a lowamount of endogenous furin.

The data indicate that the transfected fur genetic sequences are indeedtranscribed and translated making it possible to test the biologicalfunction of the fur products.

Proprotein Processing Activity of Furin

In transfection experiments with 10 μg pSVLvWF DNA, the 360 kDa pro-vWFprecursor protein and the 260 kDa mature vWF protein were found invirtually equal proportions in the conditioned medium. The formation ofmature vWF in the cells obtained by transfection is attributed to aprocessing by endogenous furin, which is expressed in COS-1 cells asshown by Northern blot analysis of mRNA isolated from COS-1 cells and byimmuno-precipitation analysis with a polyclonal rabbit anti-furin serum.

In a similar transfection experiments of COS-1 cells with 10 μgpSVLvWFgly763 DNA, it was found that pro-vWFgly763 was formed andsecreted constitutively in the culture medium as a 360 kDa protein.There was no endoproteolytic processing to mature vWF (260 kDa).

The same result was found when a cotransfection of 5 μg pSVLvWFgly763DNA and 5 μg pSVLfur DNA was carried out. No processing of pro-vWFgly763to mature vWF was observed.

On the other hand, when COS-1 cells were cotransfected with 5 μg pSVLvWFDNA and 5 μg pSVLfur DNA, a complete processing of pro-vWF to mature vWFwas found.

Materials and Methods

Molecular Cloning

pSVLfur contains a 4.1 kb full-length fur cDNA fragment, starting 117nucleotides upstream of the ATG start codon and ending 21 nucleotidesdownstream of the poly-A addition site cloned into the EcoRI site ofpSVL (Wells et al., Nucl. Acids Res. 11, 1983, 7911-7925). In pSVLfur,expression of the fur cDNA sequences is under control of the SV40 latepromoter. pMJ109 consists of a 2.2 kb SmaI/SmaI human fur cDNA fragmentmolecularly cloned into the HindIII site of plasmid pUR291; the 2.2 kbfur cDNA encompasses the carboxyterminal region of furin, which inpMJ109 is fused in phase to the β-galactosidase (β-gal) encodingsequences using the polylinker region constructed just in front of thestop codon in lacZ. pMJ119 consists of the same 2.2 kb fur cDNA fragmentbut here molecularly cloned into the SmaI site of plasmid pATH1, whichresults in the in phase fusion of the furin sequences to the first 336amino acid residues of the trpE-encoded portion of anthranilatesynthetase (336trpE-AS). Finally, in case of pEW1, a 3.5 kb BglII/EcoRIfur cDNA fragment is cloned into pGEX-3X and fused in phase toglutathion-S-transferase (GST).

Upon proper induction of protein synthesis in bacteria transformed withpMJ109, pMJ119 or pEW1, production of relatively large quantities ofβ-gal-Δfurin1 (MW 170 kDa), 336trpE-AS-Δfurin1 (MW 90 kDa) andGST-Δfurin2 (MW 100 kDa), respectively, was observed.

Preparation of Polyclonal Anti-furin Antibodies and Immunoblotting

Polyclonal anti-furin antibodies were raised in rabbits to theβ-gal-Δfurin1 hybrid protein synthesized in bacteria transformed bypMJ109. For immunizations, partially purified hybrid proteinpreparations were used. Upon size fractionation of the bacterialproteins by SDS-PAGE, the gel region containing the hybrid protein wasexcised and the protein content removed electrophoretically. Westernblotting experiments with extracts of transfected COS-1 cells wereperformed as follows. COS-1 cells transfected with 10 μg of pSVLfur DNAwere maintained 48 hr post-transfection in serum-free medium. At thistime, the cells were washed twice with 10 mM sodium phosphate (pH 7.4),0.14 M NaCl and then lyzed in "immunoprecipitation buffer (IPB)"consisting of 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 1% (v/v)Nonidet P-40, 10 mM benzamidine, 5 mM N-ethylmaleimide and 1 mMphenylmethylsulfonyl fluoride (PMSF). An aliquot of the cell extract wasrun under reducing conditions on a 8% (w/v) SDS-polyacrylamide gel and,subsequently, proteins were transferred to nitrocellulose (Schleicherand Schuell). Detection of furin was performed by incubating the blotwith the rabbit anti-furin serum described above.

DNA Transfection, Radiolabeling of Cells and ImmunoprecipitationAnalysis

Monkey kidney COS-1 cells were propagated in Iscove's modified minimalmedium, supplemented with fetal calf serum (10% v/v) and antibioticspenicillin (100 U/ml) and streptomycin (100 μg/ml)!. Twenty four hoursupon seeding, the semi-confluent cells were transfected with 20 μg ofDNA in 2 ml of Iscove's modified minimal medium, supplemented with 200μg/ml DEAE-dextran. The transfection procedure used included achloroquine chock (Luthman and Magnusson, Nucl. Acids Res. 11, 1983,1295-1308). After transfection, cells were maintained in the mediumdescribed above for 48 hours. Prior to radiolabelling, medium wasremoved and the cells incubated for 1 h in RPMI medium, lackingmethionine. Subsequently, cells were labelled for 4 hours in thepresence of ³⁵ S!methionine (50 μCi/ml, specific activity >800 Ci/mmol),followed by a chase of 14 hours with unlabelled methionine (finalconcentration 1 mM). After centrifugation for 5 min at 13,000×g, thelabelled culture media were adjusted to 1×IPB. Preclearance of the mediawas performed by incubating twice with gelatin-Sepharose and,subsequently, with preformed complexes of rabbit pre-immune serum withProtein A-Sepharose. Immunoprecipitation of radiolabelled vWF-relatedproteins was carried out by preformed complexes of an IgG preparation,derived from rabbit anti-vWF (Dakopatts, Glostrup, Denmark) with ProteinA-Sepharose. Immunoprecipitates were extensively washed with IPB andpelleted through a discontinuous 10-20% (w/v) sucrose gradient dissolvedin IPB supplemented with 0.5% desoxycholate and 10 mM Tris-HCl (pH 7.8),respectively. Immunoprecipitates were analysed under reducing conditionson a 5% SDS-polyacrylamide gel.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 12    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    -      Lys Arg Arg Thr Lys Arg    #  5 1    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 794 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    -      Met Glu Leu Arg Pro Trp Leu Leu - # Trp Val Val Ala Ala Thr Gly    Thr    #   15    -      Leu Val Leu Leu Ala Ala Asp Ala - # Gln Gly Gln Lys Val Phe Thr    Asn    #                 30    -      Thr Trp Ala Val Arg Ile Pro Gly - # Gly Pro Ala Val Ala Asn Ser    Val    #             45    -      Ala Arg Lys His Gly Phe Leu Asn - # Leu Gly Gln Ile Phe Gly Asp    Tyr    #         60    -      Tyr His Phe Trp His Arg Gly Val - # Thr Lys Arg Ser Leu Ser Pro    His    #     80    -      Arg Pro Arg His Ser Arg Leu Gln - # Arg Glu Pro Gln Val Gln Trp    Leu    #   95    -      Glu Gln Gln Val Ala Lys Arg Arg - # Thr Lys Arg Asp Val Tyr Gln    Glu    #                110    -      Pro Thr Asp Pro Lys Phe Pro Gln - # Gln Trp Tyr Leu Ser Gly Val    Thr    #            125    -      Gln Arg Asp Leu Asn Val Lys Ala - # Ala Trp Ala Gln Gly Tyr Thr    Gly    #        140    -      His Gly Ile Val Val Ser Ile Leu - # Asp Asp Gly Ile Glu Lys Asn    His    #    160    -      Pro Asp Leu Ala Gly Asn Tyr Asp - # Pro Gly Ala Ser Phe Asp Val    Asn    #   175    -      Asp Gln Asp Pro Asp Pro Gln Pro - # Arg Tyr Thr Gln Met Asn Asp    Asn    #                190    -      Arg His Gly Thr Arg Cys Ala Gly - # Glu Val Ala Ala Val Ala Asn    Asn    #            205    -      Gly Val Cys Gly Val Gly Val Ala - # Tyr Asn Ala Arg Ile Gly Gly    Val    #        220    -      Arg Met Leu Asp Gly Glu Val Thr - # Asp Ala Val Glu Ala Arg Ser    Leu    #    240    -      Gly Leu Asn Pro Asn His Ile His - # Ile Tyr Ser Ala Ser Trp Gly    Pro    #   255    -      Glu Asp Asp Gly Lys Thr Val Asp - # Gly Pro Ala Arg Leu Ala Glu    Glu    #                270    -      Ala Phe Phe Arg Gly Val Ser Gln - # Gly Arg Gly Gly Leu Gly Ser    Ile    #            285    -      Phe Val Trp Ala Ser Gly Asn Gly - # Gly Arg Glu His Asp Ser Cys    Asn    #        300    -      Cys Asp Gly Tyr Thr Asn Ser Ile - # Tyr Thr Leu Ser Ile Ser Ser    Ala    #    320    -      Thr Gln Phe Gly Asn Val Pro Trp - # Tyr Ser Glu Ala Cys Ser Ser    Thr    #   335    -      Leu Ala Thr Thr Tyr Ser Ser Gly - # Asn Gln Asn Glu Lys Gln Ile    Val    #                350    -      Thr Thr Asp Leu Arg Gln Lys Cys - # Thr Glu Ser His Thr Gly Thr    Ser    #            365    -      Ala Ser Ala Pro Leu Ala Ala Gly - # Ile Ile Ala Leu Thr Leu Glu    Ala    #        380    -      Asn Lys Asn Leu Thr Trp Arg Asp - # Met Gln His Leu Val Val Gln    Thr    #    400    -      Ser Lys Pro Ala His Leu Asn Ala - # Asn Asp Trp Ala Thr Asn Gly    Val    #   415    -      Gly Arg Lys Val Ser His Ser Tyr - # Gly Tyr Gly Leu Leu Asp Ala    Gly    #                430    -      Ala Met Val Ala Leu Ala Gln Asn - # Trp Thr Thr Val Ala Pro Gln    Arg    #            445    -      Lys Cys Ile Ile Asp Ile Leu Thr - # Glu Pro Lys Asp Ile Gly Lys    Arg    #        460    -      Leu Glu Val Arg Lys Thr Val Thr - # Ala Cys Leu Gly Glu Pro Asn    His    #    480    -      Ile Thr Arg Leu Glu His Ala Gln - # Ala Arg Leu Thr Leu Ser Tyr    Asn    #   495    -      Arg Arg Gly Asp Leu Ala Ile His - # Leu Val Ser Pro Met Gly Thr    Arg    #                510    -      Ser Thr Leu Leu Ala Ala Arg Pro - # His Asp Tyr Ser Ala Asp Gly    Phe    #            525    -      Asn Asp Trp Ala Phe Met Thr Thr - # His Ser Trp Asp Glu Asp Pro    Ser    #        540    -      Gly Glu Trp Val Leu Glu Ile Glu - # Asn Thr Ser Glu Ala Asn Asn    Tyr    #    560    -      Gly Thr Leu Thr Lys Phe Thr Leu - # Val Leu Tyr Gly Thr Ala Pro    Glu    #   575    -      Gly Leu Pro Val Pro Pro Glu Ser - # Ser Gly Cys Lys Thr Leu Thr    Ser    #                590    -      Ser Gln Ala Cys Val Val Cys Glu - # Glu Gly Phe Ser Leu His Gln    Lys    #            605    -      Ser Cys Val Gln His Cys Pro Pro - # Gly Phe Ala Pro Gln Val Leu    Asp    #        620    -      Thr His Tyr Ser Thr Glu Asn Asp - # Val Glu Thr Ile Arg Ala Ser    Val    #    640    -      Cys Ala Pro Cys His Ala Ser Cys - # Ala Thr Cys Gln Gly Pro Ala    Leu    #   655    -      Thr Asp Cys Leu Ser Cys Pro Ser - # His Ala Ser Leu Asp Pro Val    Glu    #                670    -      Gln Thr Cys Ser Arg Gln Ser Gln - # Ser Ser Arg Glu Ser Pro Pro    Gln    #            685    -      Gln Gln Pro Pro Arg Leu Pro Pro - # Glu Val Glu Ala Gly Gln Arg    Leu    #        700    -      Arg Ala Gly Leu Leu Pro Ser His - # Leu Pro Glu Val Val Ala Gly    Leu    #    720    -      Ser Cys Ala Phe Ile Val Leu Val - # Phe Val Thr Val Phe Leu Val    Leu    #   735    -      Gln Leu Arg Ser Gly Phe Ser Phe - # Arg Gly Val Lys Val Tyr Thr    Met    #                750    -      Asp Arg Gly Leu Ile Ser Tyr Lys - # Gly Leu Pro Pro Glu Ala Trp    Gln    #            765    -      Glu Glu Cys Pro Ser Asp Ser Glu - # Glu Asp Glu Gly Arg Gly Glu    Arg    #        780    -      Thr Ala Phe Ile Lys Asp Gln Ser - # Ala Leu    #    790    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 357 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    -      Asp Val Tyr Gln Glu Pro Thr Asp - # Pro Lys Phe Pro Gln Gln Trp    Tyr    #   15    -      Leu Ser Gly Val Thr Gln Arg Asp - # Leu Asn Val Lys Ala Ala Trp    Ala    #                 30    -      Gln Gly Tyr Thr Gly His Gly Ile - # Val Val Ser Ile Leu Asp Asp    Gly    #             45    -      Ile Glu Lys Asn His Pro Asp Leu - # Ala Gly Asn Tyr Asp Pro Gly    Ala    #         60    -      Ser Phe Asp Val Asn Asp Gln Asp - # Pro Asp Pro Gln Pro Arg Tyr    Thr    #     80    -      Gln Met Asn Asp Asn Arg His Gly - # Thr Arg Cys Ala Gly Glu Val    Ala    #   95    -      Ala Val Ala Asn Asn Gly Val Cys - # Gly Val Gly Val Ala Tyr Asn    Ala    #                110    -      Arg Ile Gly Gly Val Arg Met Leu - # Asp Gly Glu Val Thr Asp Ala    Val    #            125    -      Glu Ala Arg Ser Leu Gly Leu Asn - # Pro Asn His Ile His Ile Tyr    Ser    #        140    -      Ala Ser Trp Gly Pro Glu Asp Asp - # Gly Lys Thr Val Asp Gly Pro    Ala    #    160    -      Arg Leu Ala Glu Glu Ala Phe Phe - # Arg Gly Val Ser Gln Gly Arg    Gly    #   175    -      Gly Leu Gly Ser Ile Phe Val Trp - # Ala Ser Gly Asn Gly Gly Arg    Glu    #                190    -      His Asp Ser Cys Asn Cys Asp Gly - # Tyr Thr Asn Ser Ile Tyr Thr    Leu    #            205    -      Ser Ile Ser Ser Ala Thr Gln Phe - # Gly Asn Val Pro Trp Tyr Ser    Glu    #        220    -      Ala Cys Ser Ser Thr Leu Ala Thr - # Thr Tyr Ser Ser Gly Asn Gln    Asn    #    240    -      Glu Lys Gln Ile Val Thr Thr Asp - # Leu Arg Gln Lys Cys Thr Glu    Ser    #   255    -      His Thr Gly Thr Ser Ala Ser Ala - # Pro Leu Ala Ala Gly Ile Ile    Ala    #                270    -      Leu Thr Leu Glu Ala Asn Lys Asn - # Leu Thr Trp Arg Asp Met Gln    His    #            285    -      Leu Val Val Gln Thr Ser Lys Pro - # Ala His Leu Asn Ala Asn Asp    Trp    #        300    -      Ala Thr Asn Gly Val Gly Arg Lys - # Val Ser His Ser Tyr Gly Tyr    Gly    #    320    -      Leu Leu Asp Ala Gly Ala Met Val - # Ala Leu Ala Gln Asn Trp Thr    Thr    #   335    -      Val Ala Pro Gln Arg Lys Cys Ile - # Ile Asp Ile Leu Thr Glu Pro    Lys    #                350    -      Asp Ile Gly Lys Arg                 355    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 353 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    -      Arg Ile Leu Phe Asn Ile Ser Asp - # Pro Leu Phe Asp Gln Gln Trp    His    #   15    -      Leu Ile Asn Pro Asn Tyr Pro Gly - # Asn Asp Val Asn Val Thr Gly    Leu    #                 30    -      Trp Lys Glu Asn Ile Thr Gly Tyr - # Gly Val Val Ala Ala Leu Val    Asp    #             45    -      Asp Gly Leu Asp Tyr Glu Asn Glu - # Asp Leu Lys Asp Asn Phe Cys    Val    #         60    -      Glu Gly Ser Trp Asp Phe Asn Asp - # Asn Asn Pro Leu Pro Lys Pro    Arg    #     80    -      Leu Lys Asp Asp Tyr His Gly Thr - # Arg Cys Ala Gly Glu Ile Ala    Ala    #   95    -      Phe Arg Asn Asp Ile Cys Gly Val - # Gly Val Ala Tyr Asn Ser Lys    Val    #                110    -      Ser Gly Ile Arg Ile Leu Ser Gly - # Gln Ile Thr Ala Glu Asp Glu    Ala    #            125    -      Ala Ser Leu Ile Tyr Gly Leu Asp - # Val Asn Asp Ile Tyr Ser Cys    Ser    #        140    -      Trp Gly Pro Ser Asp Asp Gly Lys - # Thr Met Gln Ala Pro Asp Thr    Leu    #    160    -      Val Lys Lys Ala Ile Ile Lys Gly - # Val Thr Glu Gly Arg Asp Ala    Lys    #   175    -      Gly Ala Leu Tyr Val Phe Ala Ser - # Gly Asn Gly Gly Met Phe Gly    Asp    #                190    -      Ser Cys Asn Phe Asp Gly Tyr Thr - # Asn Ser Ile Phe Ser Ile Thr    Val    #            205    -      Gly Ala Ile Asp Trp Lys Gly Leu - # His Pro Pro Tyr Ser Glu Ser    Cys    #        220    -      Ser Ala Val Met Val Val Thr Tyr - # Ser Ser Gly Ser Gly Asn Tyr    Ile    #    240    -      Lys Thr Thr Asp Leu Asp Glu Lys - # Cys Ser Asn Thr His Gly Gly    Thr    #   255    -      Ser Ala Ala Ala Pro Leu Ala Ala - # Gly Ile Tyr Thr Leu Val Leu    Glu    #                270    -      Ala Asn Pro Asn Leu Thr Trp Arg - # Asp Val Gln Tyr Leu Ser Ile    Leu    #            285    -      Ser Ser Glu Glu Ile Asn Pro His - # Asp Gly Lys Trp Gln Asp Thr    Ala    #        300    -      Met Gly Lys Arg Tyr Ser His Thr - # Tyr Gly Phe Gly Lys Leu Asp    Ala    #    320    -      Tyr Asn Ile Val His Met Ala Lys - # Ser Trp Ile Asn Val Asn Pro    Gln    #   335    -      Gly Trp Leu Tyr Leu Pro Thr Ile - # Val Glu Lys Gln Ser Ile Ser    Asn    #                350    -      Ser    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 355 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    -      Glu Asp Lys Leu Ser Ile Asn Asp - # Pro Leu Phe Glu Arg Gln Trp    His    #   15    -      Leu Val Asn Pro Ser Phe Pro Gly - # Ser Asp Ile Asn Val Leu Asp    Leu    #                 30    -      Trp Tyr Asn Asn Ile Thr Gly Ala - # Gly Val Val Ala Ala Ile Val    Asp    #             45    -      Asp Gly Leu Asp Tyr Glu Asn Glu - # Asp Leu Lys Asp Asn Phe Cys    Ala    #         60    -      Glu Gly Ser Trp Asp Phe Asn Asp - # Asn Thr Asn Leu Pro Lys Pro    Arg    #     80    -      Leu Ser Asp Asp Tyr His Gly Thr - # Arg Cys Ala Gly Glu Ile Ala    Ala    #   95    -      Lys Lys Gly Asn Asn Phe Cys Gly - # Val Gly Val Gly Tyr Asn Ala    Lys    #                110    -      Ile Ser Gly Ile Arg Ile Leu Ser - # Gly Asp Ile Thr Thr Glu Asp    Glu    #            125    -      Ala Ala Ser Leu Ile Tyr Gly Leu - # Asp Val Asn Asp Ile Tyr Ser    Cys    #        140    -      Ser Trp Gly Pro Ala Asp Asp Gly - # Arg His Leu Gln Gly Pro Ser    Asp    #    160    -      Leu Val Lys Lys Ala Leu Val Lys - # Gly Val Thr Glu Gly Arg Asp    Ser    #   175    -      Lys Gly Ala Ile Tyr Val Phe Ala - # Ser Gly Asn Gly Gly Thr Arg    Gly    #                190    -      Asp Asn Cys Asn Tyr Asp Gly Tyr - # Thr Asn Ser Ile Tyr Ser Ile    Thr    #            205    -      Ile Gly Ala Ile Asp His Lys Asp - # Leu His Pro Pro Tyr Ser Glu    Gly    #        220    -      Cys Ser Ala Val Met Ala Val Thr - # Tyr Ser Ser Gly Ser Gly Glu    Tyr    #    240    -      Ile His Ser Ser Asp Ile Asn Gly - # Arg Cys Ser Asn Ser His Gly    Gly    #   255    -      Thr Ser Ala Ala Ala Pro Leu Ala - # Ala Gly Val Tyr Thr Leu Leu    Leu    #                270    -      Glu Ala Asn Pro Asn Leu Thr Trp - # Arg Asp Val Gln Tyr Leu Ser    Ile    #            285    -      Leu Ser Ala Val Gly Leu Glu Lys - # Asn Ala Asp Gly Asp Trp Arg    Asp    #        300    -      Ser Ala Met Gly Lys Lys Tyr Ser - # His Arg Tyr Gly Phe Gly Lys    Ile    #    320    -      Asp Ala His Lys Leu Ile Glu Met - # Ser Lys Thr Trp Glu Asn Val    Asn    #   335    -      Ala Gln Thr Trp Phe Tyr Leu Pro - # Thr Leu Tyr Val Ser Gln Ser    Thr    #                350    -      Asn Ser Thr                 355    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 278 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    -      Tyr Thr Pro Asn Asp Pro Tyr Phe - # Ser Ser Arg Gln Tyr Gly Pro    Gln    #   15    -      Lys Ile Gln Ala Pro Gln Ala Trp - # Asp Ile Ala Glu Gly Ser Gly    Ala    #                 30    -      Lys Ile Ala Ile Val Asp Thr Gly - # Val Gln Ser Asn His Pro Asp    Leu    #             45    -      Ala Gly Lys Val Val Gly Gly Trp - # Asp Phe Val Asp Asn Asp Ser    Thr    #         60    -      Pro Gln Asn Gly Asn Gly His Gly - # Thr His Cys Ala Gly Ile Ala    Ala    #     80    -      Ala Val Thr Asn Asn Ser Thr Gly - # Ile Ala Gly Thr Ala Pro Lys    Ala    #   95    -      Ser Ile Leu Ala Val Arg Val Leu - # Asp Asn Ser Gly Ser Gly Thr    Trp    #                110    -      Thr Ala Val Ala Asn Gly Ile Thr - # Tyr Ala Ala Asp Gln Gly Ala    Lys    #            125    -      Val Ile Ser Leu Ser Leu Gly Gly - # Thr Val Gly Asn Ser Gly Leu    Gln    #        140    -      Gln Ala Val Asn Tyr Ala Trp Asn - # Lys Gly Ser Val Val Val Ala    Ala    #    160    -      Ala Gly Asn Ala Gly Asn Thr Ala - # Pro Asn Tyr Pro Ala Tyr Tyr    Ser    #   175    -      Asn Ala Ile Ala Val Ala Ser Thr - # Asp Gln Asn Asp Asn Lys Ser    Ser    #                190    -      Phe Ser Thr Tyr Gly Ser Val Val - # Asp Val Ala Ala Pro Gly Ser    Trp    #            205    -      Ile Tyr Ser Thr Tyr Pro Thr Ser - # Thr Tyr Ala Ser Leu Ser Gly    Thr    #        220    -      Ser Met Ala Thr Pro His Val Ala - # Gly Val Ala Gly Leu Leu Ala    Ser    #    240    -      Gln Gly Arg Ser Ala Ser Asn Ile - # Arg Ala Ala Ile Glu Asn Thr    Ala    #   255    -      Asp Lys Ile Ser Gly Gly Thr Tyr - # Trp Ala Lys Gly Arg Val Asn    Ala    #                270    -      Tyr Lys Ala Val Gln Tyr                 275    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 274 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    -      Ala Gln Thr Val Pro Tyr Gly Ile - # Pro Leu Ile Lys Ala Asp Lys    Val    #   15    -      Gln Ala Gln Gly Phe Lys Gly Ala - # Asn Val Lys Val Ala Val Leu    Asp    #                 30    -      Thr Gly Ile Gln Ala Ser His Pro - # Asp Leu Asn Val Val Gly Gly    Ala    #             45    -      Ser Phe Val Ala Gly Glu Ala Tyr - # Asn Thr Asp Gly Asn Gly His    Gly    #         60    -      Thr His Val Ala Gly Thr Val Ala - # Ala Leu Asp Asn Thr Thr Gly    Val    #     80    -      Leu Gly Val Ala Pro Ser Val Ser - # Leu Tyr Ala Val Lys Val Leu    Asn    #   95    -      Ser Ser Gly Ser Gly Thr Tyr Ser - # Gly Ile Val Ser Gly Ile Glu    Trp    #                110    -      Ala Thr Thr Asn Gly Met Asp Val - # Ile Asn Met Ser Leu Gly Gly    Pro    #            125    -      Ser Gly Ser Thr Ala Met Lys Gln - # Ala Val Asp Asn Ala Tyr Ala    Arg    #        140    -      Gly Val Val Val Val Ala Ala Ala - # Gly Asn Ser Gly Ser Ser Gly    Asn    #    160    -      Thr Asn Thr Ile Gly Tyr Pro Ala - # Lys Tyr Asp Ser Val Ile Ala    Val    #   175    -      Gly Ala Val Asp Ser Asn Ser Asn - # Arg Ala Ser Phe Ser Ser Val    Gly    #                190    -      Ala Glu Leu Glu Val Met Ala Pro - # Gly Ala Gly Val Tyr Ser Thr    Tyr    #            205    -      Pro Thr Ser Thr Tyr Ala Thr Leu - # Asn Gly Thr Ser Met Ala Ser    Pro    #        220    -      His Val Ala Gly Ala Ala Ala Leu - # Ile Leu Ser Lys His Pro Asn    Leu    #    240    -      Ser Ala Ser Gln Val Arg Asn Arg - # Leu Ser Ser Thr Ala Thr Tyr    Leu    #   255    -      Gly Ser Ser Phe Tyr Tyr Gly Lys - # Gly Leu Ile Asn Val Glu Ala    Ala    #                270    -      Ala Gln    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 275 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    -      Ala Gln Ser Val Pro Tyr Gly Val - # Ser Gln Ile Lys Ala Pro Ala    Leu    #   15    -      His Ser Gln Gly Tyr Thr Gly Ser - # Asn Val Lys Val Ala Val Ile    Asp    #                 30    -      Ser Gly Ile Asp Ser Ser His Pro - # Asp Leu Lys Val Ala Gly Gly    Ala    #             45    -      Ser Met Val Pro Ser Glu Thr Asn - # Pro Phe Gln Asp Asn Asn Ser    His    #         60    -      Gly Thr His Val Ala Gly Thr Val - # Ala Ala Leu Asn Asn Ser Ile    Gly    #     80    -      Val Leu Gly Val Ala Pro Ser Ala - # Ser Leu Tyr Ala Val Lys Val    Leu    #   95    -      Gly Ala Asp Gly Ser Gly Gln Tyr - # Ser Trp Ile Ile Asn Gly Ile    Glu    #                110    -      Trp Ala Ile Ala Asn Asn Met Asp - # Val Ile Asn Met Ser Leu Gly    Gly    #            125    -      Pro Ser Gly Ser Ala Ala Leu Lys - # Ala Ala Val Asp Lys Ala Val    Ala    #        140    -      Ser Gly Val Val Val Val Ala Ala - # Ala Gly Asn Glu Gly Thr Ser    Gly    #    160    -      Ser Ser Ser Thr Val Gly Tyr Pro - # Gly Lys Tyr Pro Ser Val Ile    Ala    #   175    -      Val Gly Ala Val Asp Ser Ser Asn - # Gln Arg Ala Ser Phe Ser Ser    Val    #                190    -      Gly Pro Glu Leu Asp Val Met Ala - # Pro Gly Val Ser Ile Gln Ser    Thr    #            205    -      Leu Pro Gly Asn Lys Tyr Gly Ala - # Tyr Asn Gly Thr Ser Met Ala    Ser    #        220    -      Pro His Val Ala Gly Ala Ala Ala - # Leu Ile Leu Ser Lys His Pro    Asn    #    240    -      Trp Thr Asn Thr Gln Val Arg Ser - # Ser Leu Glu Asn Thr Thr Thr    Lys    #   255    -      Leu Gly Asp Ser Phe Tyr Tyr Gly - # Lys Gly Leu Ile Asn Val Gln    Ala    #                270    -      Ala Ala Gln                 275    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    -      Arg Ser Lys Arg Ser Leu    #  5 1    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #  18              TA    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #  18              TA    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    -      Arg Ser Lys Gly Ser Leu    #  5 1    __________________________________________________________________________

We claim:
 1. A process for the (micro)biological production of a proteinby culturing genetically engineered cells expressing a pro-form of theprotein as well as furin having the amino acid sequence depicted in FIG.1 or fragments thereof possessing endoprotease activity, and optionallyisolating the protein formed.
 2. A process as claimed in claim 1, inwhich genetically engineered mammalian cells are used.